The proposal builds on knowledge gained in the last project period and focusses on experimental approaches designed to understand the molecular structure and mechanism of glucosyltransferases (GTFs) expressed by oral bacteria commonly referred to as mutans streptococci. This family of oral bacteria are the most virulent microorganism associated with dental caries on smooth enamel tooth surfaces and possibly root surfaces as well. The bacterial virulence can be substantially attributed to the glucosyltransferases which synthesize extracellular long-chain glucan polysaccharides. The glucans form a scaffolding on tooth surfaces which enhances bacterial colonization. Four specific aims have been developed. AIM 1, will explore the catalytic mechanism of glucosyltransferases elements and associated loops at the enzyme active site. Also part of this aim is the application of active-site reversible. AIM 2, is designed to define high affinity inhibitors of GTF through the synthesis and screening of two forms of combinatorial libraries: an L-amino acid hexapeptide library and a unique carbohydrate-based organic library. AIM 3, is directed toward structure and function studies of the GTF glucan- binding domain which binds the glucan product and is essential for glucan synthesis. Experiments include cloning and expressing separate catalytic and glucan binding domains, and then reconstitution the enzyme noncovalently. Both homologous and heterologous reconstitution will be examined using both GTF-I and GTF-S. Catalytic parameters will be measured to assess function, and sedimentation equilibrium will be used to measure affinity between the two domains. Aim 4, is directed at structure analysis of the GTF. Two modeling strategies will be pursued to acquire information of the structure of the catalytic domain: Knowledged Based Modeling, and The method of 1D-3D profile approach to the reverse fold problem. Finally, as part of the aim, the glucan-binding domain will be prepared and provided to our collaborator for X-ray structure analysis.